Expression of sucrose phosphorylase in plants

ABSTRACT

Introducing sucrose phosphorylase activity into plants by transformation with a gene for the enzyme increases the rate of sucrose hydrolysis, leading to increased starch, oil, and/protein levels. Sucrose phosphorylase genes from Streptococcus mutans and Leuconostoc mesenteroides have been found particularly advantageous for use in the present invention. Surprisingly, in potatoes transformed to express these genes in tubers, reduced bruise discoloration susceptibility and increased uniformity of starch deposition throughout the tuber are achieved.

This application is a continuation-in-part of U.S. application Ser. No.08/596,024 filed Feb. 6, 1996, now U.S. Pat. No. 5,716,837, which is acontinuation-in-part of U.S. application Ser. No. 08/386,860 filed Feb.10, 1995 now abandoned.

Recent advances in genetic engineering have provided the requisite toolsto transform plants to contain foreign genes. It is now possible toproduce plants which have unique characteristics of agronomic and cropprocessing importance. Certainly, one such advantageous trait isenhanced starch and/or solids content and quality in various cropplants. Another is enhanced oil and protein content of seeds of variouscrop plants.

Sucrose is the carbon storage unit which is transported from the sourcetissues of most plants to the sink tissues. In sink tissues it ishydrolyzed and the components used to build other, more complex storageunits, primarily starch, protein, and oil. The hydrolysis is primarilyaccomplished by sucrose synthase which produces UDPglucose and fructose.UDPglucose is converted to glucose 1-phosphate by UDPglucosepyrophosphorylase.

The starch content of the sink tissues of various crop plants has beenincreased through the use of a gene encoding a bacterial ADPglucosepyrophosphorylase. See PCT Application WO 91/19806 (equivalent to U.S.Ser. No. 08/120,703, Kishore, incorporated herein by reference). Thisenzyme catalyzes the production of ADPglucose from glucose 1-phosphate.It has also been found that its expression during certain phases of seeddevelopment can decrease the oil content which is thought to be due tothe shunting of raw material to the starch pathway with a concomitantdecrease in its availability for oil production.

Bruising of potatoes is a phenomenon found during large-scaleproduction, handling, and storage. The bruise is seen as a dark spotprimarily in the cortex area of the tuber. Bruising can lead to loss ofquality in the tuber, lower consumer acceptance of potatoes and potatoproducts, and processing loss of tubers having excessive levels ofbruising. It has been found that potato varieties with higher starchcontent have greater susceptibility to bruising. It would be desirableto decrease the level or incidence of bruising and particularlydesirable to do so while increasing the starch content of the tuber.

A more uniform distribution of starch and solids within the potato tuberis also desirable. The pith or core of the potato generally has lowersolids content that the outer or cortex region. When longitudinal stripsare cut from the potato tuber to make french fries, the middle portionsof these strips therefore have lower solids levels than the ends andthis is especially true of strips cut from the center of the tuber.Strips with lower solids content or with regions of lower solids contentrequire longer cooking times to achieve the same degree of acceptabilityto the consumer. These longer cooking times may result in over-cookingof the higher solids strips. Longer frying times also result in greaterabsorption of fat and therefore low solids strips and those with lowersolids content regions will have a higher fat content. Higher fatcontent fries are a less nutritious food. In the manufacture of potatochips, slices are cut across the potato tuber and the non-uniformdistribution of solids can result in a fried product with overcookededges, under-cooked centers, and a higher fat content (especially in thecenter). The non-uniform distribution of solids in the potato tuber alsoresults in disproportionate losses of potato solids (from the cortex)during the peeling process.

Higher solids content is also desirable in tomato. Higher solids in theform of soluble (usually sugars and acids) and insoluble solidscontribute to processing efficiency and the yield of products such asketchup, paste, sauces, and salsa. These solids also contribute to thetaste and texture of the processed products. Higher solids alsocontribute to the improved taste of fresh tomatoes.

Sucrose phosphorylase is a microbial enzyme which catalyzes productionof glucose-1-phosphate directly from sucrose. Its activity has beenobserved in a wide range of bacterial and fungal species, and the enzymehas been isolated from a number of them (Pimentel et al., 1992; Vandammeet al., 1987). Genes for this enzyme, have been isolated fromAgrobacterium spp. (Fournier et al., 1994, and references citedtherein), Streptococcus mutans, denominated gtfA, (Russell et al., Perryet al.) and Leuconostoc mesenteroides, denominated spl (Kitao et al.,1992). Heterologous expression of the gene from S. mutans in E. coli isdisclosed in U.S. Pat. No. 4,888,170 (Curtiss, 1989), incorporatedherein by reference. The utility of the transformed microorganism is useas a vaccine against S. mutans.

It is an object of this invention to provide an improved means forincreasing starch content of various plants. It is a still furtherobject to provide a means of decreasing the sucrose content of seeds inoilseed crops resulting in a decrease in the level of undesirablecarbohydrates such as stachyose and raffinose, while increasing thecarbon available for oil and protein production. It is a still furtherobject to provide novel DNA constructs which are useful in providingsaid means. It is a still further object to provide potato tubers whichexhibit increased starch content more uniformly throughout the tuber. Itis a still further object of this invention to provide potato tuberswith a reduced susceptibility to bruising. It is a still further objectof this invention to provide improved cereal crops, such as maize, rice,wheat, and barley.

SUMMARY OF THE INVENTION

The present invention provides DNA constructs which encode a sucrosephosphorylase (SP) enzyme and which are useful in producing enhancedstarch content in plants. In another aspect of the present invention,seeds having a decreased level of sucrose and other carbohydrates, whichwill result in increased oil and protein content as a result of SPexpression are provided.

In accomplishing the foregoing, there is provided, in accordance withone aspect of the present invention, a method of modifying thecarbohydrate content of target tissues of transgenic plants, comprisingthe steps of:

-   -   (a) inserting into the genome of a plant cell a recombinant,        double-stranded DNA molecule comprising in sequence        -   (i) a promoter which functions in the cells of a target            plant tissue,        -   (ii) a structural DNA sequence that causes the production of            an RNA sequence which encodes a sucrose phosphorylase            enzyme,        -   (iii) a 3′ non-translated DNA sequence which functions in            plant cells to cause transcriptional termination and the            addition of polyadenylated nucleotides to the 3′end of the            RNA sequence;    -   (b) obtaining transformed plant cells; and    -   (c) regenerating from the transformed plant cells genetically        transformed plants.

In another aspect of the present invention there is provided arecombinant, double-stranded DNA molecule comprising in sequence

-   -   (i) a promoter which functions in the cells of a target plant        tissue,    -   (ii) a structural DNA sequence that causes the production of an        RNA sequence which encodes a sucrose phosphorylase enzyme,    -   (iii) a 3′ non-translated DNA sequence which functions in plant        cells to cause transcriptional termination and the addition of        polyadenylated nucleotides to the 3′ end of the RNA sequence.

There have also been provided, in accordance with another aspect of thepresent invention, transformed plant cells that contain DNA comprised ofthe above-mentioned elements (i), (ii), and (iii). In accordance withyet another aspect of the present invention, differentiated potato,tomato, and cereal plants are provided that have increased starchcontent in the tubers, fruit and seeds, respectively, and differentiatedoilseed crop plants are provided that have decreased sucrose andoligosaccharides containing sucrose, such as stachyose and raffinose, inthe seeds.

There have also been provided methods of increasing the starch contentin the starch production organs of plants, such as the tuber of potatoand the seed of cereals, and decreasing the sucrose levels in oilseedcrop plants, such as soybean and canola, leading to increased oil andprotein content. In carrying out the method in potato, it hasunexpectedly been found that there is a more uniform distribution ofstarch as compared between the pith and the cortex of the tuber. Inanother aspect of the invention, a method of providing potatoes having areduced susceptibility to bruising is provided.

An additional advantage of sucrose phosphorylase activity in sinktissue, such as the tuber of potato, is related to providing anincreased, novel sucrose hydrolyzing activity having a much lower Km forsucrose (1-25 mM) than that for plant sucrose hydrolyzing enzymes,sucrose synthases and invertases, which have a K_(m) in the range of50-300 mM. This advantage is important in the establishment of andstrength of such sink tissues, resulting potentially in yieldenhancement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 refers to a western blot analysis of kernels sampled at 12, 25,35 and 45 DAP.

DETAILED DESCRIPTION OF THE INVENTION

The expression of a plant gene which exists in double-stranded DNA forminvolves transcription of messenger RNA (mRNA) from one strand of theDNA by RNA polymerase enzyme, and the subsequent processing of the mRNAprimary transcript inside the nucleus. This processing involves a 3′non-translated region which adds polyadenylate nucleotides to the 3′ endof the RNA.

Transcription of DNA into mRNA is regulated by a region of DNA usuallyreferred to as the promoter. The promoter region contains a sequence ofbases that signals RNA polymerase to associate with the DNA, and toinitiate the transcription of mRNA using one of the DNA strands as atemplate to make a corresponding complimentary strand of RNA.

A number of promoters which are active in plant cells have beendescribed in the literature. These include the nopaline synthase (NOS)and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of Agrobacterium tumefaciens), the caulimoviruspromoters such as the cauliflower mosaic virus (CaMV) 19S and 35S andthe figwort mosaic virus 35S-promoters, the light-inducible promoterfrom the small subunit of ribulose-1,5-bis-phosphate carboxylase(ssRUBISCO, a very abundant plant polypeptide), and the chlorophyll a/bbinding protein gene promoter, etc. All of these promoters have beenused to create various types of DNA constructs which have been expressedin plants; see, e.g., PCT publication WO 84/02913 (Rogers et al.,Monsanto).

Promoters which are known or are found to cause transcription of DNA inplant cells can be used in the present invention. Such promoters may beobtained from a variety of sources such as plants and plant viruses andinclude, but are not limited to, the enhanced CaMV35S promoter andpromoters isolated from plant genes such as ssRUBISCO genes. Asdescribed below, it is preferred that the particular promoter selectedshould be capable of causing sufficient expression to result in theproduction of an effective amount of sucrose phosphorylase (SP) enzymeto cause the desired increase in starch content. In addition, it ispreferred to bring about expression of the SP gene in specific tissuesof the plant such as root, tuber, seed, fruit, etc. and the promoterchosen should have the desired tissue and developmental specificity.Those skilled in the art will recognize that the amount of sucrosephosphorylase needed to induce the desired increase in starch contentmay vary with the type of plant and furthermore that too much sucrosephosphorylase activity may be deleterious to the plant. Therefore,promoter function should be optimized by selecting a promoter with thedesired tissue expression capabilities and approximate promoter strengthand selecting a transformant which produces the desired sucrosephosphorylase activity in the target tissues. This selection approachfrom the pool of transformants is routinely employed in expression ofheterologous structural genes in plants since there is variation betweentransformants containing the same heterologous gene due to the site ofgene insertion within the plant genome (commonly referred to as“position effect”).

It is preferred that the promoters utilized in the double-stranded DNAmolecules of the present invention have relatively high expression intissues where the increased starch content and/or dry mater is desired,such as the tuber of the potato plant, the fruit of tomato, or seed ofmaize, wheat, rice, and barley. Expression of the double-stranded DNAmolecules of the present invention by a constitutive promoter,expressing the DNA molecule in all or most of the tissues of the plant,will be rarely preferred and may, in some instances, be detrimental toplant growth.

The class I patatin promoter has been shown to be both highly active andtuber-specific (Bevan et al., 1986; Jefferson et al., 1990). A sequenceof ˜1.0 kb portion of the tuber-specific class I patatin promoter ispreferred for tuber expression in the present invention. A number ofother genes with tuber-specific or -enhanced expression are known,including the potato tuber ADPGPP genes, both the large and smallsubunits, (Muller et al., 1990), sucrose synthase (Salanoubat andBelliard, 1987, 1989), the major tuber proteins including the 22 kdprotein complexes and proteinase inhibitors (Hannapel, 1990), thegranule bound starch synthase gene (GBSS) (Rohde et al., 1990), and theother class I and II patatins (Rocha-Sosa et al., 1989; Mignery et al.,1988). Other promoters which are contemplated to be useful in thisinvention include those that show enhanced or specific expression inpotato tubers, that are promoters normally associated with theexpression of starch biosynthetic or modification enzyme genes, or thatshow different patterns of expression within the potato tuber. Examplesof these promoters include those for the genes for the granule-bound andother starch synthases, the branching enzymes (Kossmann et al., 1991;Blennow, A. and Johansson, G., 1991; WO 92/14827; WO 92/11375),diproportionating enzyme (Takaha et al., 1993), debranching enzymes,amylases, starch phosphorylases (Nakano et al., 1989; Mori et al.,1991), pectin esterases (Ebbelaar, et al., 1993), the 40 kDglycoprotein, ubiquitin, aspartic proteinase inhibitor (Stukerlj et al.,1990), the carboxypeptidase inhibitors, tuber polyphenol oxidases(Shahar et al., 1992; GenBank® Accession Numbers M95196 and M95197),putative trypsin inhibitor and other tuber cDNAs (Stiekema et al.,1988), and for β-amylase and sporamins (from Ipomoea batatas; Yoshida etal., 1992; Ohta et al., 1991).

In addition, promoters may be identified to be tuber specific byscreening a cDNA library of potato for genes which are selectively orpreferably expressed in tubers and then determine the promoter regionsto obtain tuber selective or tuber-enhanced promoters.

Other promoters can also be used to express a sucrose phosphorylase genein specific tissues, such as seeds or fruits. β-conglycinin (also knownas the 7S protein) is one of the major storage proteins in soybean(Glycine max) (Tierney, 1987). The promoter for β-conglycinin or otherseed-specific promoters such as the napin and phaseolin promoters, canbe used to over-express an SP gene specifically in seeds. This wouldlead to a decrease in the sucrose content of the seeds, which willresult in a decrease in undesirable oligosaccharides and potentially anincrease in the oil and/or protein content, which would be desirable inseeds used for oil or protein production such sa soybean, canola,oilseed rape, sunflower, safflower, etc. The SP gene will provide moreraw material more quickly, but the plants own regulatory mechanismswill, unless influenced by other enzymes produced from heterologousgenes, direct its use in the sink tissues.

The zeins are a group of storage proteins found in maize endosperm.Genomic clones for zein genes have been isolated (Pedersen, 1982), andthe promoters from these clones, including the 15 kD, 16 kD, 19 kD, 22kD, 27 kD, and gamma genes, could also be used to express an SP gene inthe seeds of maize and other plants. Other promoters known to functionin maize include the promoters for the following genes: waxy, Brittle,Shrunken 2, Branching enzymes I and II, starch synthases, debranchingenzymes, oleosins, glutelins, and sucrose synthases. A particularlypreferred promoter for maize endosperm expression of an SP gene is thepromoter for a glutelin gene from rice, more particularly the Osgt-1promoter (Zheng et al., 1993).

If one wanted to increase oil in maize seed, rather than starch, onewould choose a promoter which causes expression of the SP gene duringoil deposition. Such a promoter would be activated during the formationof the plant embryo. Examples of promoters active during embryogenesisare the promoters from the genes for globulin 1 and the lateembryogenesis active (lea) proteins.

Examples of promoters suitable for expression of an SP gene in wheatinclude those for the genes for the ADPglucose pyrophosphorylase(ADPGPP) subunits, for the granule bound and other starch synthases, forthe branching and debranching enzymes, for the embryogenesis-abundantproteins, for the gliadins, and for the glutenins. Examples of suchpromoters in rice include those for the genes for the ADPGPP subunits,for the granule bound and other starch synthases, for the branchingenzymes, for the debranching enzymes, for sucrose synthases, and for theglutelins. A particularly preferred promoter is the promoter for riceglutelin, Osgt-1. Examples of such promoters for barley include thosefor the genes for the ADPGPP subunits, for the granule bound and otherstarch synthases, for the branching enzymes, for the debranchingenzymes, for sucrose synthases, for the hordeins, for the embryoglobulins, and the aleurone specific proteins.

The solids content of tomato fruit can be increased by expressing an SPgene behind a fruit specific promoter. The promoter from the 2A11genomic clone (Pear, 1989) will control expression of ADPglucosepyrophosphorylase in tomato fruit. The E8 promoter (Deikman, 1988) wouldalso express the SP gene in tomato fruits. In addition, promoters whichfunction during the green fruit stage of tomatoes are disclosed in PCTApplication PCTUS94/07072, filed Jun. 27, 1994, designating the U.S.,incorporated herein by reference. They are designated TFM7 and TFM9.TFM7 which is a DNA fragment, isolated from tomato, of about 2.3 kb, ofwhich 1.4 kb of the 3′ end is shown in SEQ ID NO:3. TFM9 which is a DNAfragment of about 900 bp, of which 400 bp of the 3′end is shown in SEQID NO:4.

It is also now known that potato tuber promoters will function in tomatoplants to cause fruit specific expression of an introduced gene. (SeeU.S. Ser. No. 08/344,639, Barry et al., filed Nov. 4, 1994, incorporatedherein by reference.) Such promoters include potato patatin promoters,potato ADPGPP promoters, and potato granule bound starch synthasepromoters. A particularly preferred promoter for tomato fruit expressionis the promoter for the gene encoding the small subunit of ADPGPP inpotato.

The solids content of root tissue can be increased by expressing an SPgene behind a root specific promoter. The promoter from the acidchitinase gene (Samac et al., 1990) would express the SP gene in roottissue. Expression in root tissue could also be accomplished byutilizing the root specific subdomains of the CaMV35S promoter that havebeen identified (Benfey et al., 1989).

The RNA produced by a DNA construct of the present invention may alsocontain a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the nRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs, as presented in thefollowing examples, wherein the non-translated region is derived fromthe 5′ non-translated sequence that accompanies the promoter sequence.Rather, the non-translated leader sequence can be derived from anunrelated promoter or coding sequence as discussed above.

Targeting Signal Sequences

An alternative method of increasing the rate of sucrose hydrolysis wouldbe to target the SP to the apoplast. To do so requires a signal peptideis required on the N′-terminus of the functional protein. A preferredexample of a sequence encoding such a signal sequence is a plantendoplasmic reticulum signal sequence from the PR-1B protein (Ohshima,et al., 1990). Thus the SP would be active in the apoplast and allowsucrose to be hydrolyzed extracellularly and allow for faster transportof glucose into the cell.

Another alternative is to target the SP to the vacuolar space. Targetingof the SP to the vacuole of a plant cell requires information inaddition to the signal peptide (Nakamura and Matsuoka, 1993). Aprepro-signal peptide could be fused to the amino terminus of the FT totarget the enzyme to the vacuole (Sonnewald et al., 1991).Alternatively, a carboxy terminal sequence extension could be combinedwith an ER signal sequence to target the enzyme to the vacuole.

Sucrose Phosphorylases

As used herein, the term “sucrose phosphorylase” means an enzyme whichcatalyzes a reversible conversion of sucrose and inorganic phosphate toα-D-glucose-1-phosphate and D-fructose. It may be isolated from manymicrobial sources, including Streptococcus mutans, Clostridiumpasteurianum (Vandamme et al., 1987), Pseudomonas saccharophila(Silverstein et al.), Pseudomonas putrifaciens, Pullularia pullulans,Acetobacter xylinum (Vandamme et al., 1987), Agrobacterium sp. (Fournieret al., 1994), and Leuconostoc mesenteroides.

The gene for the SP enzyme may be obtained by known methods and hasalready been isolated from several organisms, such as Agrobacterium sp.(Fournier et al., 1994) and Leuconostoc mesenteroides (Kitao et al.,1992). The gene from S. mutans has been expressed in E. coli (Robeson etal., 1983), identifying the activity as a glucosyl transferase. Theisolation and use of SP coding sequences from Streptococcus mutans(gtfA) and Leuconostoc mesenteroides (spl) is described in the Examplesbelow. These genes have been found to be particularly useful inaccordance with the methods of the present invention. Their sequencesare shown in SEQ ID NO:5 and SEQ ID NO:6, respectively. These and otherSP genes can be used for insertion into plant expression vectorssuitable for a transformation method of choice as described below.

A gene encoding SP (ORF 488) has been identified in the Ti plasmids ofAgrobacterium vitus (formerly A. tumefaciens biotype 3). Relatedsequences have been reported in the Ti plasmids of other A. tumefaciensstrains, in particular pTiC58 (Fournier et al., 1994). It is likely thata gene encoding SP may be found on all such plasmids.

Purification of the SP enzyme has been demonstrated from other bacterialand fungal sources (described above). The availability of such materialsrenders facile the subsequent cloning of the gene for this enzyme: theprotein may be used as an immunogen to raise antibodies that may be usedto identify clones in expression-based libraries such as λgt11 (Sambrooket al.); peptide sequences at the N-terminus of such proteins may beobtained by routine protein sequencing; and, following well establishedlimited proteolysis procedures, the sequences of internal regions mayalso be determined. Such sequences may be used in the design ofnucleotide probes or primers that may be used to identify the genes fromclone banks or to amplify the gene or portions of the gene from RNA,cDNA, or DNA preparations from the source organism. Detection of E. colicontaining sucrose phosphorylase clones is also possible by growth onminimal medium with sucrose as the sole carbon source (Ferretti, et al.1988).

Other microorganisms that use SP to hydrolyze sucrose can be found byassaying for organisms which can utilize sucrose as the sole carbonsource (Russell et al.). The protein can be isolated by following theenzymatic activity in the fractions using known methods. The geneencoding the protein can then be isolated as just described.

Thus, many different genes which encode an protein having sucrosephosphorylase activity may be isolated and used in the presentinvention.

Polyadenylation Signal

The 3′ non-translated region of the chimeric plant gene contains apolyadenylation signal which functions in plants to cause the additionof polyadenylate nucleotides to the 3′ end of the RNA. Examples ofsuitable 3′ regions are (1) the 3′ transcribed, non-translated regionscontaining the polyadenylated signal of Agrobacterium the tumor-inducing(Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2)plant genes like the soybean storage protein genes and the small subunitof the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. Anexample of a preferred 3′ region is that from the ssRUBISCO gene of pea,also known as the E9 3′ region.

Synthetic Gene Construction

The SP gene from Streptococcus mutans is high in A+T content, which maybe inimical to high level expression in plant cells, although as shownbelow, the gene is expressed at levels sufficient to positively affectstarch content. If desired, the gene sequence of the SP gene can bechanged without changing the protein sequence in such a manner as mayincrease expression, and thus even more positively affect starch contentin transformed plants. The rules for making the changes in the genesequence are set out in WO 90/10076 (Fischhoff et al.). A genesynthesized by following the rules set out therein may be introducedinto plants as described below and result in higher levels of expressionof the SP enzyme. This may be particularly useful in monocots such asmaize, rice, wheat, and barley.

Combinations with Other Transgenes

The effect of SP in transgenic plants can be enhanced by combining itwith other genes which positively affect starch and/or oil content. Forexample, a gene which will increase ADPglucose pyrophosphorylase(ADPGPP) activity in plants may be used in combination with an SP geneto increase starch. Such ADPGPP genes include the E. coli glgC gene andits mutant glgC16. WO 91/19806 discloses how to incorporate this geneinto many plant species in order to increase starch and/or solids.

Another gene which can be combined with SP to increase starch is a genefor sucrose phosphate synthase (SPS) which can be obtained from plants.WO 92/16631 discloses one such gene and its use in transgenic plants.

Another gene which can be combined with SP to increase oil is a gene foracetyl CoA carboxylase, which can be obtained from plants. WO 93/11243discloses one such gene.

Plant Transformation/Regeneration Plant Transformation/Regeneration

Plants which can be made to have increased polysaccharide (e.g. starch)content by practice of the present invention include, but are notlimited to, maize, wheat, rice, tomato, potato, sweet potato, peanut,barley, cotton, strawberry, raspberry, and cassava. Plants which can bemade to have modified carbohydrate content by practice of the presentinvention include, but are not limited to, maize, wheat, rice, tomato,potato, sweet potato, peanut, barley, sugarbeet, sugarcane, apple, pear,orange, grape, cotton, strawberry, raspberry, and cassava. Plants whichcan be made to have reduced bruising discoloration by practice of thepresent invention include, but are not limited to, wheat, potato, sweetpotato, barley, sugarbeet, sugarcane, apple, pear, peach, orange, grape,banana, plantain, and cassava. Plants which can be made to have improveduniform solids content by practice of the present invention include, butare not limited to potato, sweet potato, banana, plantain, and cassava.Plants which can be made to have increased yield of harvested materialby practice of the present invention include, but are not limited to,maize, wheat, rice, tomato, potato, sweet potato, peanut, barley,sugarbeet, sugarcane, apple, pear, orange, peach, banana, plantain,grape, cotton, strawberry, raspberry, and cassava. Plants which can bemade to have decreased sucrose leading to increased oil or proteincontent include soybean, maize, canola, and sunflower.

A double-stranded DNA molecule of the present invention containing an SPgene can be inserted into the genome of a plant by any suitable method.Suitable plant transformation vectors include those derived from a Tiplasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g.,by Herrera-Estrella (1983), Bevan (1984), Klee (1985) and EPOpublication 120,516 (Schilperoort et al.). In addition to planttransformation vectors derived from the Ti or root-inducing (Ri)plasmids of Agrobacterium, alternative methods can be used to insert theDNA constructs of this invention into plant cells. Such methods mayinvolve, for example, the use of liposomes, electroporation, chemicalsthat increase free DNA uptake, free DNA delivery via microprojectilebombardment, and transformation using viruses or pollen.

A plasmid expression vector, suitable for the introduction of an SP genein monocots using microprojectile bombardment is composed of thefollowing: a promoter that is specific or enhanced for expression in thestarch storage tissues in monocots, generally the endosperm, such aspromoters for the zein genes found in the maize endosperm (Pedersen etal., 1982); an intron that provides a splice site to facilitateexpression of the gene, such as the Hsp70 intron (PCT PublicationWO93/19189); and a 3′ polyadenylation sequence such as the nopalinesynthase 3′ sequence (NOS 3′; Fraley et al., 1983). This expressioncassette may be assembled on high copy replicons suitable for theproduction of large quantities of DNA.

A particularly useful Agrobacterium-based plant transformation vectorfor use in transformation of dicotyledonous plants is plasmid vectorpMON530 (Rogers, S. G., 1987). Plasmid pMON530 is a derivative ofpMON505 prepared by transferring the 2.3 kb StuI-HindIII fragment ofpMON316 (Rogers, S. G., 1987) into pMON526. Plasmid pMON526 is a simplederivative of pMON505 in which the SmaI site is removed by digestionwith XmaI, treatment with Klenow polymerase and ligation. PlasmidpMON530 retains all the properties of pMON505 and the CaMV35S-NOSexpression cassette and now contains a unique cleavage site for SmaIbetween the promoter and polyadenylation signal.

Binary vector pMON505 is a derivative of pMON200 (Rogers, S.G., 1987) inwhich the Ti plasmid homology region, LIH, has been replaced with a 3.8kb HindIII to SmaI segment of the mini RK2 plasmid, pTJS75 (Schmidhauser& Helinski, 1985). This segment contains the RK2 origin of replication,oriV, and the origin of transfer, oriT, for conjugation intoAgrobacterium using the tri-parental mating procedure (Horsch & Klee,1986). Plasmid pMON505 retains all the important features of pMON200including the synthetic multi-linker for insertion of desired DNAfragments, the chimeric NOS/NPTII′/NOS gene for kanamycin resistance inplant cells, the spectinomycin/streptomycin resistance determinant forselection in E. coli and A. tumefaciens, an intact nopaline synthasegene for facile scoring of transformants and inheritance in progeny anda pBR322 origin of replication for ease in making large amounts of thevector in E. coli. Plasmid pMON505 contains a single T-DNA borderderived from the right end of the pTiT37 nopaline-type T-DNA. Southernanalyses have shown that plasmid pMON505 and any DNA that it carries areintegrated into the plant genome, that is, the entire plasmid is theT-DNA that is inserted into the plant genome. One end of the integratedDNA is located between the right border sequence and the nopalinesynthase gene and the other end is between the border sequence and thepBR322 sequences.

Another particularly useful Ti plasmid cassette vector is pMON17227.This vector is described by Barry et al. in WO 92/04449 (correspondingto U.S. Ser. No. 07/749,611, incorporated herein by reference) andcontains a gene encoding an enzyme conferring glyphosate resistance(denominated CP4) which is an excellent selection marker gene for manyplants, including potato and tomato. The gene is fused to theArabidopsis EPSPS chloroplast transit peptide (CTP2) and expressed fromthe FMV promoter as described therein.

When adequate numbers of cells (or protoplasts) containing the SP geneor cDNA are obtained, the cells (or protoplasts) are regenerated intowhole plants. Choice of methodology for the regeneration step is notcritical, with suitable protocols being available for hosts fromLeguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot,celery, parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.),Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice,maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), variousfloral crops, such as sunflower, and nut-bearing trees, such as almonds,cashews, walnuts, and pecans. See, e.g., Ammirato, 1984; Shimamoto,1989; Fromm, 1990; Vasil, 1990; Vasil, 1992; Hayashimoto, 1989;Shimamoto, 1989; and Datta, 1990.

The following examples are provided to better elucidate the practice ofthe present invention and should not be interpreted in any way to limitthe scope of the present invention. Those skilled in the art willrecognize that various modifications, truncations, etc. can be made tothe methods and genes described herein while not departing from thespirit and scope of the present invention.

All basic DNA manipulations and genetic techniques described in theExamples provided herein, unless otherwise stated, such as PCR, agaroseelectrophoresis, restriction digests, ligations, E. colitransformations, Western blots etc. were performed by standard protocolsas described in Sambrook et al. (1989) and/or Maniatis et al. (1982).

EXAMPLES Example 1

A sucrose phosphorylase gene, gtfA, was generated by PCR amplificationfrom Streptococcus mutans cells. The gene was amplified using the 5′oligonucleotide:

-   5CCCGGATCCATGGCAATTACAAATAAAAC (SEQ ID NO:1) and the 3′    oligonucleotide:-   5′GGGGAGCTCACTCGAAGCTTATTGTTTGATCATTTTCTG (SEQ ID NO:2)

The PCR cycling conditions were as follows: 94° C., 3′;55° C., 2′;72°C., 2′ (5 cycles);94° C. 1′;55° C. 2′;72° C. 2′ (30 cycles). The 1462 bpPCR product was purified using the GeneClean purification system(Bio101, Vista, Calif.), digested with BamHI and SacI, and ligated intothe BamHI and SacI sites of pUC119. The ligated DNA was transformed intoJM101 and a blue-white screen was used to identify colonies for plasmidpreparation and restriction digestion. Digestion with HindIII was usedto screen for transformants containing the gtfA gene. Clones withcorrect restriction patterns were screened for phenotypic expression bythe ability to utilize sucrose as sole carbon source as follows: cloneswere transformed into a gal-E. coli strain, SK1592, and grown on minimalmedium containing raffinose (which is taken up and hydrolyzed togalactose and sucrose) and an active clone was identified and namedpMON17353.

An expression cassette was constructed to allow for constitutiveexpression of gtfA in plants. A fragment containing the enhanced 35Spromoter (Kay, R. 1987), the Nopaline synthase 3′ region (Bevan, M.1984), and the pUC vector backbone was prepared from pMON999 (Rogers etal., 1987a) by restriction digestion with BglII and SacI. A fragmentcontaining the gtfA coding region was prepared from pMON17353 byrestriction digestion with BamHI and SacI. The correct fragments wereseparated by agarose gel electrophoresis and purified by the GeneCleanprocedure. The fragments were ligated, transformed into E. coli JM 101,and putative recombinant plasmids were screened by restriction digestionwith NotI. One clone was identified and named pMON17359.

A second expression cassette was constructed to direct expression ofgtfA to the potato tuber. A fragment containing the patatin 1.0 promoter(described above), the Nopaline synthase 3′ region, and the pUC vectorbackbone was prepared from an intermediate vector by restrictiondigestion with BamHI and SacI. An expression cassette was alsoconstructed to direct expression of gtfA to the tomato fruit. A fragmentcontaining the TFM7 promoter, the Nopaline synthase 3′ region, and thepUC vector backbone was prepared from pMON16987 (PCT ApplicationPCTUS94/07072, filed Jun. 27, 1994), which is derived from pMON999 butcontains the TFM7 promoter, by restriction digestion with BglII andSacI. The correct fragments were separated by agarose gelelectrophoresis and purified by the GeneClean procedure. These fragmentswere each ligated to the BamHI and SacI fragment from pMON17353.Transformation and screening of clones were as described above. Cloneswere designated as correct and named pMON17356 (Pat 1.0/gtfA/NOS) andpMON17389 (TFM7/gtfA/NOS).

A third expression cassette was constructed to direct expression of gtfAto the potato tuber using a 3.5 kb promoter of patatin. The patatin 3.5promoter was obtained from the plasmid pBI240.7 (Bevan et al., 1986).The majority of the 3.5 promoter was excised from pBI240.7, from theHindIII site (at˜3500) to the XbaI site at −337, and combined with theremainder of the promoter, from the XbaI site to a BglII site at +22(formerly a DraI site), in a triple ligation into a vector whichprovided a BglII site to form pMON17280. An intermediate vector wasprepared by digestion of pMON17353 with BamHI/SacI and insertion of thefragment into pBS. This vector was then digested with EcoRI and SacI.pMON17280 was digested with EcoRI and SacI resulted in a fragmentcontaining the patatin 3.5 promoter, the Nopaline synthase 3′ region,and the pUC vector backbone. The correct sized fragments were obtainedby agarose gel electrophoresis and the GeneClean procedure. Thefragments were ligated, transformed into E. coli JM101, and screened byrestriction digestion with HindIII. One clone was designated as correctand named pMON17495.

In pMON17356, pMON17359, pMON17389, and pMON17495, the promoter, gtfAgene and the Nos 3′ region can be isolated on a NotI restrictionfragment. These fragments can then be inserted into a unique NotI siteof either vector pMON17227 (described above) or pMON17320 to constructglyphosate selectable plant transformation vectors. pMON17320 is apMON17227 derivative which also contains a Patatin 1.0/CTP1-glgC16cassette. The CTP1-glgC16 fusion encodes a modified ADPglucosepyrophosphorylase as described by Kishore in WO 91/19806. A vector wasalso constructed for tomato expression of gtfA by combining the gtfAgene and 3′ region from pMON17356 with the ˜2.0 kp potato smallADPglucose pyrophosphorylase subunit gene promoter (See U.S. Ser. No.08/344,639, Barry et al., filed Nov. 4, 1994, incorporated herein byreference.) in a plant transformation vector to form pMON17486.

The vector DNA is prepared by digestion with NotI followed by treatmentwith calf intestinal alkaline phosphatase (CIAP). The gtfA containingfragments are prepared by digestion with NotI, agarose gelelectrophoresis and purification with GeneClean. Vector and insert DNAis ligated, transformed into the E. coli strain LE392, and transformantswere screened by restriction digestion to identify clones containing thegtfA expression cassettes. Clones in which transcription from the gtfAcassette is in the same direction as transcription from the selectablemarker were designated as correct and named pMON17357 (FMV/CP4/E9,Pat1.0/gtfA/NOS), pMON17358 (Pat1.0/CTP1-glgC16/E9, Pat1.0/gtfA/NOS,FMV/CP4/E9), pMON17360 (FMV/CP4/E9, E35S/gtfA/NOS), pMON17390(FMV/CP4/E9, TFM7/gtfA/NOS), pMON17392 (Pat1.0/CTP1-glgC16/E9,TFM7/gtfA/NOS, FMV/CP4/E9), and pMON17496 (FMV/CP4/E9, Pat3.5/gtfA/NOS).

A transformation vector was constructed to direct expression of gtfA inmaize seed. A fragment containing the glutelin promoter Osgt-1, Hsp70intron (described above). Nopaline synthase 3′ region, kanamycinresistance, and pUC backbone was prepared by restriction digestion,agarose gel electrophoresis, and GeneClean. A fragment containing thegtfA coding region was prepared from pMON17359 by restriction digestionwith NcoI and NotI. The fragments were ligated, transformed and screenedby restriction digestion. A correct clone was identified and namedpMON24502 (Osgt1/Hsp70/gtfA/NOS).

A transformation vector was constructed to direct expression of gtfA inseeds of oilseed crops. A BamHI-EcoRI fragment of pMON17353 was ligatedinto the BglII-EcoRI sites of an intermediate vector to give pMON26104which placed the gtfA gene behind the 7s promoter (discussed above) andused the E9 3′ terminator sequence. A NotI fragment containing the FMVpromoter, the fusion of the CTP2 and glyphosate resistance gene and aNos 3′ sequence, was ligated into the NotI site of pMON26104 to givepMON26106, a double border plant transformation vector with both of thecassettes in the same orientation.

Example 2

The vector pMON17357 was transformed into Russet Burbank potato callusfollowing the method described by Barry et al. in WO 94/28149 forglyphosate selection of transformed lines. A number of lines wereobtained and evaluated in field tests. The results of this test areshown in Table 1. As can be seen therein, several lines were identifiedas containing higher starch levels (measured as total solids) and someof those had decreased bruising.

TABLE 1 Bruising Line Solids (%) Index Identification Mean Mean Control21.9 3.399  1 22.9 3.798  3 22.2 3.479  4 23.3 2.899  6 21.8 2.798  822.7 2.979 11 21.6 2.968 12 22.0 3.383 14 22.3 3.218 15 22.7 2.979 1722.3 3.394 18 21.7 3.394 19 22.4 3.213 22 22.7 3.503

Tubers from twelve lines were tested for any change in the distributionof starch between the pith or cortex. This was accomplished by peelingthe tubers, cutting them into strips resembling french fries, andmeasuring solids using a brine flotation comparison test. The averagesolids level for strips from the pith was subtracted from the averagesolids level for strips from the cortex. Thus a difference in solidswhich is less than that for the control (4.61% in this test) is anindication of more uniform distribution of starch in the tuber, which ishighly desirable. The results are shown in Table 2. As can be seen, thedifference in solids between the cortex and the pith was reduced in tenof the twelve lines.

TABLE 2 Line Solids Difference (%) Control 4.61  3 4.53  4 4.16  6 3.57 8 3.20 11 3.94 12 4.39 14 4.67 15 3.56 17 2.61 18 3.79 19 5.19 22 3.92

Five of these lines were tested the next year in the field, four of themin multiple locations. (Line number 8 was tested in only one location).The absolute increase in solids in those five lines, indicating anincrease in starch content, was again demonstrated in each line. Theresults are shown in Table 3.

TABLE 3 Line Solids increase  1 0.256  4 0.856  8 2.61 15 0.211 22 0.162

Example 3

Expression of gtfA in corn introduces a novel catalytic activity whichmay facilitate sucrose import into the endosperm by creating a steeperconcentration gradient and conserve energy since the equivalent of onemole of ATP is normally required to convert sucrose to a hexose plus ahexose phosphate. The vector pMON24502 has been introduced into maizecells by microprojectile bombardment using two different types ofembryogenic callus tissue for transformation. It was cotransformed witheither (1) pMON19476 which contains a selection cassette of the enhanced35S promoter, the Hsp 70 intron, the NPTII coding sequence for kanamycinresistance, and the nos 3′ sequence or (2) pMON19336 which contains twoselection cassettes for glyphosate resistance, each using the rice actinpromoter and the Hsp70 intron, but one uses a gene encoding glyphosateoxidase and one uses the CP4 glyphosate resistance gene.

(1) Immature maize embryos (H99 genotype) were isolated as described inEP 586 355 A2. Embryogenic callus was obtained by culturing the immatureembryos for about two weeks on the medium described by Duncan et al.(1985), called Medium D. After 2 weeks, callus (Type I) is obtained andis maintained by subculturing every 2-3 weeks onto fresh Medium D.Approximately four hours prior to bombardment, actively growing callus(mid subculture cycle) is placed on Medium D with added mannitol andsorbitol for osmotic pretreatment. Approximately 16-24 hours afterbombardment with particles coated with pMON24502 and pMON19476, thetissue is placed on Medium D without mannitol or sorbitol. Approximatelytwo days later, the tissue is transferred onto Medium D containingparomomycin. Resistant tissue is transferred to fresh Medium D withparomomycin at approximately three week intervals. Plant regeneration isaccomplished on Medium D with 6-benzylaminopurine (without dicamba) fora 3-6 day “pulse”, followed by placement on MS medium without hormones.

(2) Type II callus, derived from immature embryos of the “Hi-II”genotype, is used by following the method of Dennehy et al., 1994. TypeII callus was pretreated on N6 1-100-25 medium containing 0.4 Mmannitol+sorbitol (0.2 M of each) for four hours prior to bombardmentwith pMON24502 and pMON19336 and left on this same medium for 16 to 24hours after bombardment. The tissue was then transferred to N6 1-100-25medium without added mannitol or sorbitol. Selection was accomplishedusing 1-3 mM glyphosate in N6 1-0-25 medium (containing no casaminoacids).

Fertile maize plants have been obtained by each method and their seedstested. Production of the GtfA protein was confirmed by Western blotanalysis using goat antibody raised against E. coli-expressed gtfA. Ofthe 16 lines screened, 9 express GftA, at approximately 0.05 to 0.5% ofthe total cellular protein. The starch biosynthetic rate in maizeendosperm tissue expressing GftA (sucrose phosphorylase) was measured invitro using a sugar feeding assay which has been described previously(Felker, et al., 1990). Field grown plants were screened by PCR toidentify the positive and negative segregants. Positive and control earsfrom two GftA transformed lines (Knowl and De) were harvested at 20 to22 days post pollination, at the time of linear grain fill. Endospermsections were recovered and were fed ¹⁴C-sucrose at concentrations of 50and 200 mM. The 200 mM concentration is the most physiologicallyrelevant but due to the lower Km of GftA for sucrose than the endogenousenzymes, the lower concentration (50 mM) was used to improve thelikelihood of measuring an effect from GftA. Time points were taken atone and two hours after feeding ¹⁴C and the radioactivity incorporatedinto the starch fraction was determined. The results with the two lines,are summarized in the following Table (data reported as average countsincorporated into starch fraction):

TABLE 4A Feeding with 50 mM sucrose time of sampling control Knowl 1 hr9033 20895 2 hr 15947 26695 De 1 hr 10909 10860 2 hr 19193 24284

TABLE 4B Feeding with 200 mM sucrose time of sampling control Knowl 1 hr9880 12980 2 hr 11175 21407 De 1 hr 7703  7471 2 hr 11038 13007The results demonstrate that corn endosperm tissues expressing GtfA canproduce starch at a more rapid rate (two-fold) than controls. Thedifferences in starch rate are more apparent at the lower substrateconcentrations, potentially due to the differences in substrate kineticsbetween GtfA and the endogenous sucrose synthase. Differences were alsonoted when comparing the effects in the lines De and Knowl, with Knowldisplaying a more positive effect. GtfA protein was very high in Knowl,in the range of 0.5% of the total protein, whereas GtfA levels in Dewere in the range of 0.05%. The differences in starch biosynthetic ratesare likely a function of GtfA expression levels.

Example 4

The vector pMON26106 has been introduced into canola and soybean callusvia Agrobacterium transformation (Hinchee et al.). After selection oftransformed cells using glyphosate and regeneration into whole plants,the seeds set by those plants will be analyzed.

Example 5

The vector pMON24502 has been introduced into wheat cells bymicroprojectile bombardment. Immature wheat embryos were isolated asdescribed by Vasil et al. (1993). Embryogenic callus was obtained byculturing the immature embryos for 4 to 7 days, on a modified MS mediumcomprising about 40 g/l maltose and about 2 mg/l 2,4-D. The callus wassubjected to bombardment with microprojectiles coated with pMON24502 anda plasmid containing a bialophos resistance gene. One day afterbombardment the immature embryos were transferred to a growth mediumcontaining the selective agent bialaphos. After seven days on the growthand selective medium the immature embryo-derived callus was removed to ashoot-producing medium (modified MS medium no 2,4-D) containingbialophos and grown for 28-40 days. A PCR assay will be done to confirmthat the gtfA gene is present in the shoots. Shoots containing the gtfAgene will be rooted and taken to soil. When transformed plants arerecovered and grown to maturity, their seeds will exhibit increasedstarch levels.

Example 6

The vector pMON24502 may be introduced into rice cells bymicroprojectile bombardment. Upon regeneration and selection,transformed plants will be assayed for expression of the gtfA gene andthose plants demonstrating high expression will be grown to maturity.The seeds of the mature plants will exhibit increased starch levels.

Example 7

In order to evaluate the kinetic properties of two sucrose phosphorylaseenzymes, the sucrose phosphorylase genes from Streptococcus mutans andLeuconostoc mesenteroides were separately cloned into pET15 expressionvectors so that they could be overexpressed in E. coli.

The Leuconostoc mesenteroides gene sequence was obtained from Genbank(Accession Number D90314; Kitao et al., 1992) and nucleotide primerswith homology to the 5′ and 3′ ends were designed for PCR amplification.Leuconostoc mesenteroides chromosomal DNA was extracted and the spl genewas amplified by PCR using the 5′ oligonucleotide:

-   -   5′ GGGGAGATCTAACCATGGAAATTCAAAACAAAGC 3′ (SEQ ID NO:7)    -   and the 3′ oligonucleotide:    -   5′ GGGGGAGCTCATTAGTTCTGAGTCAAATTATC 3′ (SEQ ID NO: 8).        The 1.5 kb PCR product was gel purified and digested with NcoI        and SacI. The resulting restriction fragment was gel purified        and ligated into the NcoI/SacI backbone of an E. coli expression        vector, pMON5723, to form a vector construct which was used for        transformation of component E. coli cells. The pMON5723 vector        contains the E. coli recA promoter and the T7 gene 10 leader        (G10L) sequences which enable high levels of expression in E.        coli (Wong et al., 1988).

A 1491 bp NcoI/BamHI fragment containing the spl gene, encoding theLeuconostoc mesenteroides sucrose phosphorylase enzyme, was isolatedfrom this vector construct using restriction digestion, gel purified,and cloned into the BamHI/NcoI backbone of the expression vector pET15b(obtained from Novagen), following manufacturer's instructions,resulting in the plasmid pMON21633. Similarly, a 1461bp NcoI/BamHIfragment containing the gtfA gene coding for the Streptococcus mutanssucrose phosphorylase, was cloned into the expression vector pET15b,resulting in the plasmid pMON21634. The resulting plasmids, pMON21633and pMON21634, were transformed into ‘subcloning efficiency’-DH5αcompetent cells (obtained from Gibco BRL), serving as a non-expressionhost to obtain establishment of the correct plasmids. Subsequently,these plasmid constructs were used for the transformation of competentBL21 (DE3) cells, a host strain provided with the pET system, to obtainhigh levels of expression. All DNA manipulations, cell transformation,selection etc. were performed according to the manufacturer's directions(Novagen).

For the purification of sucrose phosphorylase (Spl or GtfA), colonies ofcells containing the correct plasmid construct were picked and used togrow liquid cultures (under Ampicillin selection), induced with IPTG(all purification steps were conducted at 4° C.). After growing aninduced cell culture, the bacterial cell pellet was spun down andsuspended in Buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5% glyceroland 40 μl/ml protease inhibitor cocktail, purchased from BoehringerMannheim Cat.#1-697-498)and cells were lysed using a French Press. Thecell lysate was clarified by centrifugation at 15,000 rpm for 10minutes. Ammonium sulfate was added to the supernatant to reach 30%saturation, and the resultant mixture was then applied onto aphenyl-Sepharose 4B column which was pre-equilibrated with Buffer B (10mM Tris-HCl, pH 8.0, 1 mM DTT and 1 mM EDTA) plus 20% (w/v) ammoniumsulfate. The sucrose phosphorylase was eluted from the column with alinearly decreased ammonium sulfate gradient concentration from 20% to0% in Buffer B. The fractions containing active sucrose phosphorylasewere collected and pooled. Combined proteins were concentrated byprecipitation with 70% saturation of ammonium sulfate followed byresuspension of the protein pellet in a small volume (2 or 4 ml) ofBuffer C (10 mM Tris-HCl, 1 mM EDTA and 10% Glycerol). The sample wasdialyzed in 2 liters of Buffer C two to three times (about 2 h for each)to completely remove ammonium sulfate prior to further purification. Thedialyzed sample was injected into a Mono Q column which waspre-equilibrated with Buffer C. The sucrose phosphorylase was elutedfrom the column with a linear gradient of NaCl from 0 to 0.4 M in BufferC. Sucrose phosphorylase active fractions were collected and analyzed bySDS-PAGE which indicated that the sucrose phosphorylase was at least 90%pure (at least 98% pure for Spl; at least 95% pure for GtfA), withmolecular masses around 53 kDa.

Spl and GtfA activities were measured at 25° C. using a continuousspectrophotometric assay in which the production of glucose-1-phosphatefrom sucrose and inorganic phosphate is coupled to the production ofNADP in the presence of phosphoglucomutase and glucose-6-phosphatedehydrogenase. The assay reaction mixture (1 ml final volume) contained50 mM Hepes-NaOH pH 7.0, 50 mM sucrose, 5 mM MgCl2, 1 mM NADP, 20 mM Kpimonobasic pH 7.0 (50 mM for GtfA), 8 IU phosphoglucomutase, 2 IUglucose-6-phosphate dehydrogenase and appropriate amounts of Spl orGtfA. Production of NADPH was monitored spectrophotometrically at 340nm. One unit of Spl or GtfA activity was defined as the amount of theenzyme which caused the reduction of 1 μmol of NADP per minute under theabove conditions. A control reaction in which inorganic phosphate wasomitted from the reaction mixture was always conducted for each Spl orGtfA assay.

Enzyme kinetic analysis yielded results shown in the following table.Unexpectedly, sucrose phosphorylase from S. mutans had a Vmax about 9times lower than that of the enzyme from L. mesenteroides. In addition,the Km(sucrose) of the S. mutans enzyme was about 10 times lower thanthat of the L. mesenteroides enzyme.

Km_((sucrose)) Km_((Pi)) Vmax I_(0.5(Pi)) (mM) (mM) (U/mg) (mM) S.mutans SP 0.33 15.3 8.5 ˜70 L. mesenteroides SP 3.39 4.88 71.3 ˜55

Example 8

To further characterize the sucrose phosphorylases from S. mutans and L.mesenteroides, plasmid constructs containing the spl or the gtfA geneswere made for protoplast transformation. A 1461 bp NcoI/BamHI fragment,containing the gtfA gene was isolated by restriction digestion, gelpurified, and ligated to a 4646 bp BamHI/NcoI vector fragment containingthe enhanced 35S promoter (e35S; Kay et al., 1987), the HSP70 intron(U.S. Pat. No. 5,593,874), the Nopaline synthase 3′ region (Fraley etal., 1983), and a pUC vector derived backbone. A vector was thuscreated, containing the expression cassette [p-e35S/HSP70/gtfA/NOS3′].After ligation, the plasmid constructs were transformed into competentDH5α cells, and the transformed cells were plated out onAmpicillin-containing medium. From the colonies that were formed,positive clones were identified by restriction digest. In a similarexperiment a 1491bp NcoI/BamHI fragment, containing the spl gene, wasisolated by restriction digestion, gel purified, and ligated to the same4646bp BamHI/NcoI vector fragment (described above). After ligation,transformation of competent cells, and identification of positiveclones, the resulting plasmid constructs were used for corn leafprotoplast transformation. Corn leaf protoplast transformation wasperformed essentially as described by Sheen J., 1991, with somemodifications to the protocol. The protoplast culture media was MSFromm+0.6M mannitol (Fromm et al., 1987). Protoplast transformationswere done in triplicate for both the gtfA and spl constructs.

Extraction buffer containing 100 mM Hepes pH 7.5, 1 mM EDTA, 5 mM DTT, 1mM Benzamidine, 5% glycerol, and 40 μl/ml protease inhibitor cocktailsolution at 1 tablet/2 ml (Boehringer Mannheim Cat.#1-697-498), wasadded to harvested protoplasts and the mixture was ground about 1-2minutes. The protoplast homogenates were transferred into eppendorftubes and spun at 14,000 rpm. To desalt the supernatant, spin columns(Boehringer Quick Spin Columns Cat. # 100973) were washed 3-5 times withdouble deionized H₂O, then once with 1 ml of extraction buffer. Thebuffer was removed from the spin column by centrifugation at 1400 rpmfor 2 minutes. The protoplast extract was applied onto each column, spunat 1400 rpm for 2 minutes, and the passthrough was collected. 40 μlprotease inhibitor cocktail stock solution (1 tablet/2 ml) was addedinto every ml of desalted extract. The desalted samples were used forenzyme assays.

Spl or GtfA activity was measured at 35° C. using a continuousspectrophotometric assay in which the production of glucose-1-phosphatefrom sucrose and inorganic phosphate is coupled to the production ofNADP in the presence of phosphoglucomutase and glucose-6-phosphatedehydrogenase. The assay reaction mixture (1 ml final volume) contains50 mM Hepes-NaOH pH 7.0, 100 mM sucrose, 5 mM MgCl2, 5 mM NaF, 1 mM NADPand 50 mM Kpi monobasic pH 7.0, 796 μl of reaction mix was mixed with acoupled enzyme mix (8 IU phosphoglucomutase, 2 IU glucose-6-phosphatedehydrogenase final concentrations) and 100 μl of the desaltedprotoplast extract. Sucrose (100 mM final concentration) was added toinitiate the reaction. Production of NADPH was monitoredspectrophotometrically at 340 nm. One unit of Spl or GtfA activity wasdefined as the amount of enzyme which caused the reduction of 1 μmol ofNADP per minute under the above conditions. A control reaction in whichinorganic phosphate was omitted from the reaction mixture was conductedfor each assay. Under these reaction conditions (50 mM P_(i)) GtfA hasoptimal activity (100%), but Spl only reaches about 80% of its optimalactivity.

The following results were obtained:

Average Core Protein Units/mg Units/mg Protoplasts AU/min Units/ml mg/mlprotein protein gtfA-1 0.0058 0.047 0.186 0.02526 0.03154 gtfA-2 0.00620.0498 0.109 0.04567 gtfA-3 0.0039 0.0039 0.313 0.0132 spl-1 0.02450.1967 0.082 0.23984 0.21753 spl-2 0.0197 0.1582 0.082 0.19295 spl-30.0172 0.1385 0.063 0.21979 control 0.0005 0.0038 0.228 0.00166

As seen from the Table, Spl exhibits about a 7-fold increase in enzymeactivity compared to GtfA in corn protoplasts.

Example 9

A vector was constructed for use in expressing spl and other sucrosephosphorylases in core, wheat or rice endosperm. For this, a 1588 bpBamHI/SmaI DNA fragment, containing the T7 gene 10 leader sequence(G10L) and the spl gene sequence, was isolated and ligated to an 8555 bpSmaI/BamHI vector fragment containing the HSP70 intron, the riceglutelin promoter Osgt-1 (Zheng et al., 1993), a pUC vector-derivedbackbone, and an expression cassette for the neomycin phosphotransferasetype II gene (to confer Kanamycin resistance). From the resultingplasmid vector, a 10131 bp SstI/SmaI fragment was isolated and ligatedto a 287 bp SmaI/SstI fragment, isolated from pMON999 (Rogers et al.,1987a), containing the NOS3′ polyadenylation sequence. This created aplasmid vector, from which a 7831 bp NotI expression cassette[p-osgt-1/HSP70 intron/G10L/spl/NOS3′] was isolated and ligated into theNotI site of pMON30460, a monocot transformation vector, to form theplant transformation vector pMON17588. pMON30460 contains an expressioncassette for the selectable marker neomycin phosphotransferase type IIgene (KAN) [P-35S/NPTII/NOS3′] and a unique NotI site for cloning thegene of interest. The final vector (pMON17588) was constructed so thatthe gene of interest and the selectable marker gene could be easilycloned in the same orientation. A vector fragment containing theexpression cassettes for these gene sequences can be excised from thebacterial selector (Kan) and ori, gel purified, and used for planttransformation.

Transgenic maize plants were produced using microprojectile bombardment,a procedure well-known in the art (Fromm et al., 1990; Gordon-Kamm etal., 1990; Walters et al., 1992). Embryogenic callus initiated fromimmature maize embryos was used as a target tissue. Plasmid DNA at 1mg/ml in TE buffer was precipitated onto M10 tungsten particles using acalcium chloride/spermidine procedure, essentially as described by Kleinet al. (1988). In addition to the gene of interest, the plasmids alsocontained the neomycin phosphotransferase II gene (nptII) driven by the35S promoter from Cauliflower Mosaic Virus. The embryogenic callustarget tissue was pre-treated on culture medium osmotically bufferedwith 0.2M mannitol plus 0.2M sorbitol for approximately four hours priorto bombardment (Vain et al., 1993). Tissue was bombarded two times withthe DNA-coated tungsten particles using the gunpowder version of theBioRad Particle Delivery System (PDS) 1000 device. Approximately 16hours following bombardment, the tissue was subcultured onto a medium ofthe same composition except that it contained no mannitol or sorbitol,and it contained an appropriate aminoglycoside antibiotic, such as G418″to select for those cells which contained and expressed the 35S/nptIIgene. Actively growing tissue sectors were transferred to freshselective medium approximately every 3 weeks. About 3 months afterbombardment, plants were regenerated from surviving embryogenic callusessentially as described by Duncan and Widholm (1988).

Kernels (R1 seed) were harvested from R0 maize plants, transformed withthe construct pMON17588, which contains the spl gene linked to theendosperm-specific rice glutelin promoter and the HSP70 intron. Thekernels were screened by both sucrose phosphorylase activity assays andWestern blot analysis. For this, kernels were harvested at 20 DAP (daysafter pollination) from one plant per line and approximately 8 kernelsper plant. Each sample was initially screened for Spl activity. Sampleswith no activity were indentified as non-expressors and those showingSpl enzyme activity were further screened by Spl western blot analysis.A significant correlation was observed between Spl enzyme activity andSpl protein levels, i.e., all the samples with high Spl enzyme activityalso showed high Spl protein levels by Western blot analysis. Out of 64plants that were screened, 31 showed both Spl enzyme activity and Splprotein by these analyses.

Example 10

Maize lines expressing the gtfA transgene [Osgt1/HSP70/gtfA/NOS3′]described in Example 1 (derived from pMON24502) up to approximately 0.5%of the total protein levels have been identified and one homozygous R2line “Majorie” was evaluated by an in vitro culture system as describedby Cheikh and Jones, 1995 and Jones et al., 1981.

Analysis of the developmental profile of maize kernels expressing thegtfA transgene, line “Majorie”, compared to that of the negative(non-transgenic) controls, demonstrated higher dry weight (−15%) atphysiological maturity (45 DAP, days after pollination) in the gtfAexpressors.

Dry Weight (mg/kernel) Negative control 105.8 ± 310 gtfA expressors120.3 ± 2.55

As shown in FIG. 1, Western analysis of kernels sampled at 12, 25, 35and 45 DAP revealed that elevated GtfA levels were detectable between 12and 45 DAP.

Analysis of kernel developmental variation in nonstructural carbohydratelevels showed significantly higher levels of fructose and starch andlower levels of sucrose during the period of gtfA expression, but nosignificant pattern of variation in glucose content (see Tables below).These patterns of variations reflect the activity and the effect of thegtfA transgene in the maize endosperm.

Changes in starch levels of maize kernels expressing gtfA as compared tothose of negative controls are shown in the following Table:

Kernel Starch (mg/g dry weight) Negative Kernel Starch (mg/grain) DAPControl gtfA positive Negative Control gtfA positive 20  416.9 ± 11.9 424.5 ± 12.7 16.42 ± 0.91 17.53 ± 0.87 24 524.5 ± 2.6  558.4 ± 12.434.10 ± 1.17 32.94 ± 0.46 28 523.4 ± 9.3 553.0 ± 8.7 41.38 ± 2.96 40.55± 0.92 35 533.6 ± 9.3 576.0 ± 3.9 45.57 ± 2.56 55.20 ± 0.84 40 540.5 ±9.7 592.5 ± 9.1 57.11 ± 1.37 69.68 ± 2.16

Changes in soluble carbohydrate levels of maize kernels expressing gtfAas compared to those of negative controls were also evaluated and theresults are shown in the following Table:

Glucose Fructose Sucrose DAP (mg/kernel) (mg/kernel) (mg/kernel) 12Negative 2.53 ± 0.15 1.87 ± 0.10 1.36 ± 0.12 Positive 2.15 ± 0.06 1.60 ±0.03 1.25 ± 0.15 20 Negative 1.68 ± 0.14 0.93 ± 0.09 1.49 ± 0.20Positive 1.76 ± 0.09 1.17 ± 0.05 0.89 ± 0.06 24 Negative 2.11 ± 0.090.98 ± 0.32 1.58 ± 0.12 Positive 1.68 ± 0.05 1.08 ± 0.04 0.53 ± 0.05 28Negative 1.80 ± 0.12 0.68 ± 0.04 1.31 ± 0.10 Positive 1.93 ± 0.06 1.20 ±0.37 0.58 ± 0.07 35 Negative 1.40 ± 0.08 0.49 ± 0.33 1.47 ± 0.10Positive 2.03 ± 0.09 1.09 ± 0.03 0.70 ± 0.09 40 Negative 0.69 ± 0.120.30 ± 0.03 1.41 ± 0.07 Positive 0.63 ± 0.05 0.30 ± 0.03 1.45 ± 0.06

All publications and patents mentioned in this specification are hereinincorporated by reference as if each individual publication or patentwas specifically and individually stated to be incorporated byreference.

From the foregoing, it will be seen that this invention is one welladapted to attain all the ends and objects hereinabove set forthtogether with advantages which are obvious and which are inherent to theinvention.

It will be understood that certain features and subcombinations are ofutility and may be employed without reference to other features andsubcombinations. This is contemplated by and is within the scope of theclaims.

Since many possible embodiments may be made of the invention withoutdeparting from the scope thereof, it is to be understood that all matterherein set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

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1. A method of producing a transgenic plant, comprising the steps of:(a) stably transforming into the genome of a plant cell a recombinant,double-stranded DNA molecule comprising: (i) a promoter which functionsin cells of target plant tissue, (ii) a structural DNA sequence obtainedfrom a bacterium of the genus Leuconostoc that causes the production ofan RNA sequence which encodes a sucrose phosphorylase enzyme, (iii) a 3′non-translated DNA sequence which functions in plant cells to causetranscriptional termination and the addition of polyadenylatednucleotides to the 3′ end of the RNA sequence; (b) selecting fortransformed plant cells; and (c) regenerating from said transformedplant cells a genetically transformed plant, the genome of whichcontains said recombinant, double-stranded DNA molecule of step (a),wherein said genetically transformed plant exhibits a property one ormore properties selected from the group consisting of containing amodified carbohydrate content; increased polysaccharide content;enhanced yield of harvested material; improved uniformity of thedistribution of solids; increased oil content; increased proteincontent; and reduced susceptibility to bruising discoloration.
 2. Themethod of claim 1, wherein said DNA sequence is obtained fromLeuconostoc mesenteroides.
 3. The method of claim 2, wherein said DNAsequence comprises the sequence shown in SEQ ID NO:6.
 4. The method ofclaim 1, wherein said property is containing a modified carbohydratecontent.
 5. The method of claim 4, wherein said modified carbohydratecontent is an increase in solids content.
 6. The method of claim 5,wherein said genetically transformed plant is selected from the groupconsisting of potato and tomato.
 7. The method of claim 1, wherein saidproperty is improved uniformity of the distribution of solids.
 8. Themethod of claim 7, wherein said genetically transformed plant isselected from the group consisting of potato and sweet potato.
 9. Themethod of claim 1, wherein said property is reduced susceptibility tobruising discoloration.
 10. The method of claim 9, wherein saidgenetically transformed plant is selected from the group consisting ofpotato, banana, apple, wheat, grape, and peach.
 11. The method of claim1, wherein said property is increased polysaccharide content.
 12. Themethod of claim 11, wherein said genetically transformed plant isselected from the group consisting of maize, wheat, rice, tomato,potato, sweet potato, peanut, barley, cotton, strawberry, raspberry, andcassava.
 13. The method of claim 1, wherein said property is enhancedyield of harvested material.
 14. The method of claim 13, wherein saidgenetically transformed plant is selected from the group consisting ofmaize, wheat, rice, tomato, potato, sweet potato, peanut, barley,sugarbeet, sugarcane, apple, pear, orange, peach, grape, cotton,strawberry, raspberry, and cassava.
 15. The method of claim 1, whereinsaid plant cell is selected from the group consisting of a potato plantcell, a maize plant cell, a rice plant cell, a wheat plant cell, atomato plant cell, a barley plant cell, a sugarbeet plant cell, asweetpotato plant cell, a peanut plant cell, a sugarcane plant cell, agrape plant cell, a pear plant cell, an apple plant cell, an orangeplant cell, a cassava plant cell, a banana plant cell, a plantain plantcell, a cotton plant cell, a strawberry plant cell, a raspberry plantcell, and a peach plant cell.
 16. The method of claim 15, wherein saidplant cell is a potato plant cell.
 17. The method of claim 15, whereinsaid plant cell is a maize plant cell.
 18. The method of claim 15,wherein said plant cell is selected from the group consisting of a wheatplant cell, a barley plant cell, a rice plant cell, and a tomato plantcell.
 19. A recombinant, double-stranded DNA molecule comprising insequence: (a) a promoter which functions in cells of target planttissue; (b) a structural DNA sequence obtained from a bacterium of thegenus Leuconostoc that causes the production of an RNA sequence whichencodes a sucrose phosphorylase enzyme; and (c) a 3′ non-translatedregion which functions in plant cells to cause transcriptionaltermination and the addition of polyadenylated nucleotides to the 3′ endof the RNA sequence.
 20. The DNA molecule of claim 19, wherein said DNAsequence is obtained from Leuconostoc mesenteroides.
 21. The DNAmolecule of claim 20, wherein said DNA sequence comprises the sequenceshown in SEQ ID NO:6.
 22. The DNA molecule of claim 19, wherein saidpromoter is selected from the group consisting of a zein promoter, apatatin promoter, a rice glutelin promoter, the soybean 7s promoter, apromoter of a subunit of ADPglucose pyrophosphorylase, the TFM7promoter, and the TFM9 promoter.
 23. A transformed plant cell comprisinga recombinant, doublestranded DNA molecule comprising in sequence: (a) apromoter which functions in said plant cell; (b) a structural DNAsequence obtained from a bacterium of the genus Leuconostoc that causesthe production of an RNA sequence which encodes a sucrose phosphorylaseenzyme; and (c) a 3′ non-translated region which functions in plantcells to cause transcriptional termination and the addition ofpolyadenylated nucleotides to the 3′ end of the RNA sequence.
 24. Theplant cell of claim 23, wherein said DNA sequence is obtained fromLeuconostoc mesenteroides.
 25. The plant cell of claim 24, wherein saidDNA sequence comprises the sequence shown in SEQ ID NO:6.
 26. The plantcell of claim 23, wherein said promoter is selected from the groupconsisting of a zein promoter, a patatin promoter, a rice glutelinpromoter, the soybean 7s promoter, a promoter of a subunit of ADPglucosepyrophosphorylase, the TFM7 promoter, and the TFM9 promoter.
 27. Theplant cell of claim 23, wherein said plant cell is selected from thegroup consisting of a potato plant cell, a maize plant cell, a riceplant cell, a wheat plant cell, a tomato plant cell, a barley plantcell, a sugarbeet plant cell, a sweetpotato plant cell, a peanut plantcell, a sugarcane plant cell, a grape plant cell, a pear plant cell, anapple plant cell, an orange plant cell, a cassava plant cell, a bananaplant cell, a plantain plant cell, and a peach plant cell.
 28. Atransformed plant comprising a recombinant, doublestranded DNA moleculecomprising in sequence: (a) a promoter which functions in said plantcell; (b) a structural DNA sequence obtained from a bacterium of thegenus Leuconostoc that causes the production of an RNA sequence whichencodes a sucrose phosphorylase enzyme; and (c) a 3′ non-translatedregion which functions in plant cells to cause transcriptionaltermination and the addition of polyadenylated nucleotides to the 3′ endof the RNA sequence.
 29. The plant of claim 28, wherein said DNAsequence is obtained from Leuconostoc mesenteroides.
 30. The plant cellof claims claim 29, wherein said DNA sequence comprises the sequenceshown in SEQ ID NO:6.
 31. The plant of claim 28, wherein said promoteris selected from the group consisting of a zein promoter, a patatinpromoter, a rice glutelin promoter, the soybean 7s promoter, a promoterof a subunit of ADPglucose pyrophosphorylase, the TFM7 promoter, and theTFM9 promoter.
 32. The plant of claim 28, wherein said plant is selectedfrom the group consisting of a potato plant, a maize plant, a riceplant, a wheat plant, a tomato plant, a barley plant, a sugarbeet plant,a sweetpotato plant, a peanut plant, a sugarcane plant, a grape plant, apear plant, an apple plant, an orange plant, a cassava plant, a bananaplant, a plantain plant, and a peach plant.
 33. The method of claim 1,wherein the polysaccharide is starch.
 34. The method of claim 11,wherein the polysaccharide is starch.
 35. The method of claim 1, whereinthe property is increased oil content.
 36. The method of claim 1,wherein the property is increased protein content.
 37. The method ofclaim 1, wherein said plant cell is a canola plant cell.
 38. The methodof claim 1, wherein said plant cell is a soybean plant cell.
 39. Theplant cell of claim 23, wherein said plant cell is a canola plant cell.40. The plant cell of claim 23, wherein said plant cell is a soybeanplant cell.
 41. The plant of claim 28, wherein said plant is a canolaplant.
 42. The plant of claim 28, wherein said plant is a soybean plant.43. The method of claim 1, wherein the one or more properties aremodified carbohydrate content and increased oil content.
 44. The methodof claim 1, wherein the one or more properties are modified carbohydratecontent and increased protein content.